RESUMO
The formation of insoluble complexes between enzymes and polyelectrolytes is a suitable technique for isolating these biomolecules from natural sources, because it is a simple and rapid technique that allows the concentration of the protein. This technique can be used in most purification protocols at the beginning of the downstream process. The aim of this investigation is to isolate papain from Carica papaya latex by precipitation of insoluble complexes between this enzyme and poly (vinyl sulfonate). The papain-poly (vinyl sulfonate) complex was insoluble at pH lower than 6, with a PVS/PAP stoichiometric ratio of 1:279. Ionic strength affected the complex formation. The presence of the polymer increased the enzymatic activity and protected the enzyme from autodegradation. The optimal conditions for the formation of insoluble papain-polyelectrolyte complex formation were applied to C. papaya latex and a high recovery was obtained (around 86%) and a purification factor around 2. This method can be applied as an isolation method of papain from C. papaya latex or as a first step in a larger purification strategy.
Assuntos
Carica/química , Látex/química , Papaína/isolamento & purificação , Polivinil/química , Ácidos Sulfônicos/química , Precipitação Química , Concentração de Íons de Hidrogênio , Cinética , Concentração Osmolar , Papaína/química , Papaína/metabolismo , Polivinil/metabolismo , Solubilidade , Ácidos Sulfônicos/metabolismoRESUMO
The partitioning patterns of papain (PAP) and bromelain (BR), two well-known cysteine-proteases, in polyethyleneglycol/sodium citrate aqueous two-phase systems (ATPSs) were determined. Polyethyleneglycols of different molecular weight (600, 1000, 2000, 4600 and 8000) were assayed. Thermodynamic characterization of partitioning process, spectroscopy measurements and computational calculations of protein surface properties were also carried out in order to explain their differential partitioning behavior. PAP was observed to be displaced to the salt-enriched phase in all the assayed systems with partition coefficients (KpPAP) values between 0.2 and 0.9, while BR exhibited a high affinity for the polymer phase in systems formed by PEGs of low molecular weight (600 and 1000) with partition coefficients (KpBR) values close to 3. KpBR values resulted higher than KpPAP in all the cases. This difference could be assigned neither to the charge nor to the size of the partitioned biomolecules since PAP and BR possess similar molecular weight (23,000) and isoelectric point (9.60). The presence of highly exposed tryptophans and positively charged residues (Lys, Arg and His) in BR molecule would be responsible for a charge transfer interaction between PEG and the protein and, therefore, the uneven distribution of BR in these systems.
Assuntos
Bromelaínas/química , Extração Líquido-Líquido , Papaína/química , Bromelaínas/metabolismo , Dicroísmo Circular , Ativação Enzimática , Papaína/metabolismo , Polietilenoglicóis/química , TermodinâmicaRESUMO
Affinity partitioning combines the partitioning behavior of biological macromolecules in aqueous two-phase systems with the principle of biorecognition. Among the numerous substances that have been evaluated as ligands, the reactive dyes constitute a group of low cost textile dyes which have proved to act as biomimetic ligands for many enzymes. The ability of reactive yellow 2 (RY2) to interact with trypsin (TRP) and chymotrypsin (ChTRP) and its behavior in aqueous two-phase systems formed by polyethylene glycol (PEG) and sodium citrate (NaCit) - were investigated. Different variables such as PEG molecular weight, tie line length and dye concentration were analyzed. RY2 showed to bind specifically to both TRP and ChTRP with affinity constants near to 10(3)M(-1). Its partition equilibrium is practically displaced to the top phase in systems formed by PEG of different molecular weight. Addition of this dye to PEG 8000/NaCit systems until a final concentration of 0.196% (w/w) induced an increase in TRP and ChTRP partition coefficients of at least 2 times over that in the absence of the ligand. These findings demonstrate that RY2 fulfils all the requirements to be considered as an affinity ligand in aqueous two-phase partitioning of TRP and ChTRP.
Assuntos
Compostos Azo/química , Extração Líquido-Líquido/métodos , Pâncreas/enzimologia , Serina Endopeptidases/isolamento & purificação , Triazinas/química , Animais , Compostos Azo/farmacologia , Soluções Tampão , Bovinos , Quimotripsina/antagonistas & inibidores , Quimotripsina/isolamento & purificação , Citratos/química , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinética , Ligantes , Polietilenoglicóis/química , Serina Endopeptidases/química , Citrato de Sódio , Triazinas/farmacologia , Tripsina/isolamento & purificaçãoRESUMO
La intoxicación con la Karwinskia humboldtiana presenta un cuadro neurológico similar a la poliomielitis, al síndrome de Guillain-Barré y a otras polirradiculoneuritis con las que suele confundirse. El objetivo de este trabajo fue conocer la frecuencia de esta intoxicación, mediante el antecedente de la ingestión del fruto y la detección de toxinas en sangre por cromatografía en capa fina. Se recibieron 154 muestras de casos con parálisis fláccida aguda procedentes de 18 estados de la República. Se corroboró el antecedente de ingestión en 56 de ellos y la detección fue positiva en 50 de los mismos. En 98 pacientes no hubo antecedentes de ingestión y la detección fue negativa en 95 de ellos. Se obtuvo con este método una sensibilidad de 89 por ciento y una especificidad de 96.9 por ciento
Intoxication produced by Karwinskia humboldtiana presents a neurological picture similar to that of poliomyelitis,Guillain-Barre syndrome or other polyradiculoneuritis with which it is frequently confused. The purpose of this paper is to report the frequency of this intoxication, by means of the antecedent of ingestion of the fruit and the detection of toxins in blood using a thin layer chromatography method. One hundred fifty four samples of cases with acute flaccid paralysis from 18 states of the country were received. The antecedent of ingestion in 56 of them was corroborated and the detection was positive in 50 of these. In 98 patients there was not antecedent of ingestion and detection was negative in 95 of them. We estimated that the sensibility and specificity of detection method are 89% and 96.9% respectively.
Assuntos
Humanos , Masculino , Feminino , Lactente , Pré-Escolar , Criança , Adolescente , Idoso , /toxicidade , Diagnóstico Diferencial , Doenças Transmitidas por Alimentos/diagnóstico , Doenças Transmitidas por Alimentos/epidemiologia , Paralisia/etiologiaRESUMO
La ingestión del fruto maduro de la Karwinskia humboldtiana, arbusto comúnmente conocido como tullidora o coyotillo, provoca una intoxicación descrita en la bibliografía como una parálisis fláccida, simétrica de los miembros inferiores, progresiva y ascendente, que en casos graves puede causar parálisis bulbar y muerte. Se presenta el caso de una familia en la que diez de sus trece miembros ingirieron accidentalmente el fruto de la tullidora; tres fallecieron: el padre y dos hijas. Además, se describe por primera vez, la determinación de las toxinas en sangre por medio de cromatografía en capa fina. Este método resulta útil para el diagnóstico diferencial con otras polirradiculoneuritis, v.gr. poliomielitis y Síndrome de Guillain-Barre